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United States Patent |
6,159,445
|
Klaveness
,   et al.
|
December 12, 2000
|
Light imaging contrast agents
Abstract
The present invention relates to the use of particulate materials as
contrast agents in vivo light imaging.
Inventors:
|
Klaveness; Jo (Oslo, NO);
Fuglass; Bjorn (Oslo, NO);
Rongved; P.ang.al (Oslo, NO);
Johannesen; Edvin (Oslo, NO);
Henrichs; Paul Mark (Wayne, PA);
Heinrich; Wolfgang Hans (Wayne, PA);
Bacon; Edward Richard (Wayne, PA);
Toner; John Luke (Wayne, PA);
McIntire; Gregory Lynn (Wayne, PA);
Desai; Vinay C. (Pheonixville, PA)
|
Assignee:
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Nycomed Imaging AS (NO)
|
Appl. No.:
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984771 |
Filed:
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December 4, 1997 |
Current U.S. Class: |
424/9.6; 424/9.1; 600/314; 600/317; 600/473; 600/476 |
Intern'l Class: |
A61K 049/00 |
Field of Search: |
424/9.6,9.4,9.52,9.61,450,9.1
600/310,314,317,473,476
|
References Cited
U.S. Patent Documents
3806592 | Apr., 1974 | Imondi | 424/7.
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5093106 | Mar., 1992 | Dzbanovsky et al. | 424/7.
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5140463 | Aug., 1992 | Yoo et al. | 359/559.
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5437274 | Aug., 1995 | Khoobehi et al. | 128/633.
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5460800 | Oct., 1995 | Walters | 424/9.
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5498421 | Mar., 1996 | Grinstaff et al. | 424/450.
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5505932 | Apr., 1996 | Grinstaff et al. | 424/9.
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5508021 | Apr., 1996 | Grinstaff et al. | 424/9.
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5512268 | Apr., 1996 | Grinstaff et al. | 424/9.
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Foreign Patent Documents |
0 446 028 A2 | Jun., 1991 | EP.
| |
0 446 028 A3 | Jun., 1991 | EP.
| |
2 184 015 | Jun., 1987 | GB.
| |
Other References
Bjerknes, et al., "Human Leukocyte Phagocytosis of Zymosan Particles
Measured by Flow Cytometry", Acta path.microbiol.immunol.scand. Sect. C,
91:341-348, 1983.
Cheng, et al., "The Production and Evaluation of Contrast-Carrying
Liposomes Made with an Automatic High-Pressure System", Investigative
Radiology, 22:47-55, 1997.
Jain, "Barriers to Drug Delivery in Solid Tumors", Scientific American, pp.
58-65, Jul. 1994.
|
Primary Examiner: Dees; Jose G.
Assistant Examiner: Hartley; Michael G.
Attorney, Agent or Firm: Fish & Richardson P.C.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No.
08/875,645 filed Jul. 31 1997, which is a 371 of International patent
application no. PCT/GB96/00222 filed Feb. 2, 1996.
Claims
We claim:
1. A method of generating an image of the human or non-human animal body by
in vivo diagnostic light imaging, characterized in that a physiologically
tolerable, chromophore- and fluorophore-free particulate contrast agent is
administered into said body and an image of at least part of said body
containing said particulate agent is generated.
2. A method as claimed in claim 1 wherein said image is a spatial image.
3. A method as claimed in claim 1 wherein said image is a temporal image.
4. A method as claimed in claim 1 wherein said image is generated from
light transmitting through at least part of said body.
5. A method as claimed in claim 1 wherein said image is generated from
light reflected from at least part of said body.
6. A method as claimed in claim 1 wherein said image is of vasculature in
said body.
7. A method as claimed claim 1 wherein said image is of a phagocytic organ
in said body.
8. A method as claimed in claim 1 wherein light is emitted and detected
endoscopically.
9. A method as claimed in claim 1 wherein said body is irradiated with
monochromatic light.
10. A method as claimed in claim 1 wherein the particle size of said
particulate agent is from .lambda./4.PI. to .lambda./.PI., where .lambda.
is the wavelength of said monochromatic light.
11. A method as claimed in claim 10 wherein said particle size is about
.lambda./2.PI..
12. A method as claimed in claim 1 wherein said agent comprises
gas-microbubbles.
13. A method as claimed in claim 1 wherein said agent comprises solid or
liquid particles.
14. A method as claimed in claim 1 wherein said agent comprises liposomes.
15. A method as claimed in claim 1 wherein said particulate agent comprises
micelles.
16. A method as claimed in claim 1 wherein said image is generated by a
light imaging technique selected from confocal scanning laser microscopy,
optical coherence tomography, and laser doppler and laser speckle
techniques.
17. A method as claimed in claim 16 wherein said body is illuminated with
light of a wavelength in the range 600 to 1300 nm and said image is
generated using detected scattered light of a wavelength in the range 600
to 1300 nm.
18. A method as claimed in claim 17 wherein the particles of said
particulate agent have a mean particle size in the range 600 to 1300 nm.
19. A method as claimed in claim 18 wherein the coefficient of variation of
the particle size of said particles is less than 10%.
20. A method as claimed in claim 16 wherein said image is an image of a
part of said body no more than 1 mm below an exposed or endoscopically
accessed surface thereof.
21. A method as claimed in claim 20 wherein said image is an image of part
of the skin.
22. A method as claimed in claim 20 wherein said image is an image of a
part of said body no more than 1 mm below a surgically exposed surface.
23. A method as claimed in claim 16 wherein said particles are selected
from polymer particles, and iodinated organic compound particles.
Description
FIELD OF THE INVENTION
The present invention relates to the use of particulate contrast agents in
various diagnostic imaging techniques based on light, more particularly to
particulate light imaging contrast agents.
BACKGROUND OF THE INVENTION
Contrast agents are employed to effect image enhancement in a variety of
fields of diagnostic imaging, the most important of these being X-ray,
magnetic resonance imaging (MRI), ultrasound imaging and nuclear medicine.
Other medical imaging modalities in development or in clinical use today
include magnetic source imaging and applied potential tomography. The
history of development of X-ray contrast agents is almost 100 years old.
The X-ray contrast agents in clinical use today include various
water-soluble iodinated aromatic compounds comprising three or six iodine
atoms per molecule. The compounds can be charged (in the form of a
physiologically acceptable salt) or non-ionic. The most popular agents
today are non-ionic substances because extensive studies have proven that
non-ionic agents are much safer than ionics. This has to do with the
osmotic loading of the patient. In addition to water-soluble iodinated
agents, barium sulphate is still frequently used for X-ray examination of
the gastrointestinal system. Several water-insoluble or particulate agents
have been suggested as parenteral X-ray contrast agents, mainly for liver
or lymphatic system imaging. Typical particulate X-ray contrast agents for
parenteral administration include for example suspensions of solid
iodinated particles, suspensions of liposomes containing water-soluble
iodinated agents or emulsions of iodinated oils.
The current MRI contrast agents generally comprise paramagnetic substances
or substances containing particles (hereinafter "magnetic particles")
exhibiting ferromagnetic, ferrimagnetic or superparamagnetic behaviour.
Paramagnetic MRI contrast agents can for example be transition metal
chelates and lanthanide chelates like Mn EDTA and Gd DTPA. Today, several
gadolinium based agents are in clinical use; including for example Gd DTPA
(Magnevist.RTM.), Gd DTPA-BMA (Omniscan.RTM.), Gd DOTA (Dotarem.RTM.) and
Gd HPDO3A (Prohance.RTM.). Several particulate paramagnetic agents have
been suggested for liver MRI diagnosis; for example suspensions of
liposomes containing paramagnetic chelates and suspensions of paramagnetic
solid particles like for example gadolinium starch microspheres. Magnetic
particles proposed for use as MR contrast agents are water-insoluble
substances such as Fe.sub.3 O.sub.4 or .delta.-Fe.sub.2 O.sub.3 optionally
provided with a coating or carrier matrix. Such substances are very active
MR contrast agents and are administered in the form of a physiologically
acceptable suspension.
Contrast agents for ultrasound contrast media generally comprise
suspensions of free or encapsulated gas bubbles. The gas can be any
acceptable gas for example air, nitrogen or a perfluorocarbon. Typical
encapsulation materials are carbohydrate matrices (e.g. Echovist.RTM. and
Levovist.RTM.), proteins (e.g. Albunex.RTM.), lipid matrials like
phospholipids (gas-containing liposomes) and synthetic polymers.
Markers for diagnostic nuclear medicine like scintigraphy generally
comprise radioactive elements like for example technetium (99m) and indium
(III), presented in the form of a chelate complex, whilst
lymphoscintigraphy is carried out with radiolabelled technetium sulphur
colloids and technetium oxide colloids.
The term "light imaging" used here includes a wide area of applications,
all of which utilize an illumination source in the UV, visible or IR
regions of the electromagnetic spectrum. In light imaging, the light,
which is transmitted through, scattered by or reflected (or re-emitted in
the case of fluorescence) from the body, is detected and an image is
directly or indirectly generated. Light may interact with matter to change
its direction of propagation without significantly altering its energy.
This process is called elastic scattering. Elastic scattering of light by
soft tissues is associated with microscopic variations in the tissue
dielectric constant. The probability that light of a given wavelength
(.lambda.) will be scattered per unit length of travel in tissue is termed
the (linear) scattering coefficient .mu..sub.s. The scattering coefficient
of soft tissue in an optical window of approx. 600-1300 nm ranges from
10.sup.1 -10.sup.3 cm.sup.-1 and decreases as 1/.lambda.. In this range
.mu..sub.s >>.mu..sub.a (the absorption coefficient) and although
.mu..sub.s (and the total attenuation) is very large, forward scattering
gives rise to substantial penetration of light into tissue. Ballistic
light is light that has travelled through a region of tissue without being
scattered. Quasi-ballistic light ("snake" light) is scattered light that
has maintained approximately the same direction of travel. The effective
penetration depth shows a slow increase or is essentially constant with
increasing wavelengths above 630 nm (although a slight dip is observed at
the water absorption peak at 975 nm). The scattering coefficient shows
only a gradual decrease with increasing wavelength.
Light that is scattered can either be randomly dispersed (isotropic) or can
scatter in a particular direction with minimum dispersion (anisotropic)
away from the site of scattering. For convenience and mathematical
modelling purposes, scattering in tissue is assumed to occur at discrete,
independent scattering centers ("particles"). In scattering from such
"particles", the scattering coefficient and the mean cosine of scatter
(phase function) depend on the difference in refractive index between the
particle and its surrounding medium and on the ratio of particle size to
wavelength. Scattering of light by particles that are smaller than the
wavelength of the incident light is called Rayleigh scattering. This
scattering varies as 1/.lambda..sup.4 and the scattering is roughly
isotropic. Scattering of light by particles comparable to or larger than
the wavelength of light is referred to as Mie scattering. This scattering
varies as 1/.lambda. and the scattering is anisotropic (forward peaked).
In the visible/near-IR where most measurements have been made, the
observed scattering in tissue is consistent with Mie-like scattering by
particles of micron scale: e.g. cells and major organelles.
Since the scattering coefficient is so large for light wavelengths in the
optical window (600-1300 nm), the average distance travelled by a photon
before a scattering event occurs is only 10-100 .mu.m. This suggests that
photons that penetrate any significant distance into tissue encounter
multiple scattering events. The ballistic component of light that has
travelled several centimeters through tissue is exceedingly small.
Multiple scattering in tissue means that the true optical path length is
much greater than the physical distance between the light input and output
sites. The scattering acts, therefore, to diffuse light in tissue
(diffuse-transmission and -reflection). The difficulty that multiple
scattering presents to imaging is three-fold: (i) light that has been
randomized due to multiple scattering has lost signal information and
contributes noise to the image (scattering increases noise); (ii)
scattering keeps light within tissue for a greater period of time,
increasing the probability for absorption, so less light transmits through
tissue for detection (scattering decreases signal); and (iii) the
determination of physical properties of tissue (or contrast media) such as
concentration that could be obtained from the Beer-Lambert law is
complicated since the true optical path length due to scattering is
difficult to determine (scattering complicates the quantification of light
interactions in tissue). However, although light cannot penetrate more
than a few tens of microns in tissue without being scattered, the large
value of the mean cosine of scattering indicates that a significant
fraction of photons in an incident beam may undergo a large number of
scatters without being deviated far from the original optical axis, and as
such can contribute in creating an image. As a result, it can be possible
to perform imaging on tissue despite the predominance of scatter, if the
noise component can be rejected and the quasi-ballistic component of the
light can be detected.
The most interesting wavelengths for light imaging techniques are in the
approximate range of 600-1300 nm. These wavelengths have the ability to
penetrate relatively deeply into living tissue without absorption by
natural substances and furthermore are harmless to the human body.
However, for optical analysis of surface structures or diagnosis of
diseases very close to the body surface or body cavity surfaces or lumens,
UV light and visible light below 600 nm wavelength can also be used.
Light can also be used in therapy; thus for example in Photodynamic Therapy
(PDT) photons are absorbed and the energy is transformed into heat and/or
photochemical reactions which can be used in cancer therapy.
The main methods of light imaging today include simple transillumination,
various tomographic techniques, fluorescence imaging, and hybrid methods
that involve irradiation with or detection of other forms of radiation or
energy in conjunction with irradiation with or detection of light (such as
photoacoustic or acousto-optical). These methods take advantage of either
transmitted, scattered or emitted (fluorescence) photons or a combination
of these effects. The present invention relates to contrast agents for any
of these and further imaging methods based on any form of light.
There is today great interest in development of new equipment for imaging
based on light. Interesting methods are especially the various types of
tomographic techniques in development especially in Japan. As scientific
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There are several patent publications which relate to light imaging
technology and to the use of various dyes in light imaging: a labeling
fluorescent dye comprising hydroxy aluminium 2,3-pyrido cyanide in JP
4,320,456 (Hitachi Chem), therapeutic and diagnostic agent for tumors
containing fluorescent labelled phthalocyanine pigment in JP 4288 022
(Hitachi Chem), detection of cancer tissue using visible native
luminescence in U.S. Pat. No. 4,930,516 (Alfano R. et al.), method and
apparatus for detection of cancer tissue using native fluorescence in U.S.
Pat. No. 5,131,398 (Alfano, R. et al.), improvements in diagnosis by means
of fluorescenct light emmision from tissue in WO 90/10219
(Andersson-Engels, S. et al.), fluorescent porphyrin and fluorescent
phthalocyanine-polyethylene glycol, polyol, and saccharide derivatives as
fluorescent probes in WO91/18006 (Diatron Corp), method of imaging a
random medium in U.S. Pat. No. 5,137,355 (State Univ. of New York),
tetrapyrrole therapeutic agents in U.S. Pat. No. 5,066,274 (Nippon
Petrochemicals), tetrapyrrole polyaminomonocarboxylic acid in therapeutic
agents in U.S. Pat. No. 4,977,177 (Nippon Petrochemicals), tetrapyrrole
aminocarboxylic acids in U.S. Pat. No. 5,004,811 (Nippon Petrochemicals),
porphyrins and cancer treatment in U.S. Pat. No. 5,162,519 (Efamol
Holdings), dihydroporphyrins and method of treating tumors susceptible to
necrosis in U.S. Pat. No. 4,837,221 (Efamol), parenterally administered
zinc phthalocyanide compounds in form of liposome dispersion containing
synthetic phospholipids in EP 451 103 (CIBA Geigy), apparatus and method
for detecting tumors in U.S. Pat. No. 4,515,165 (Energy Conversion
Devices), time and frequency domain spectroscopy determining hypoxia in
WO92/13598 (Nim Inc), phthalocyanatopolyethylene glycol and phthalocyanato
saccharides as fluorescent digoxin reagent in WO 91/18007 (Diatron),
fluorometer in U.S. Pat. No. 4,877,965 (Diatron), fiberoptic fluorescence
spectrometer in WO 90/00035 (Yale Univ.), tissue oxygen measuring system
in EP 502,270 (Hamamatsu Photonics), method for determining bilirubin
concentration from skin reflectance in U.S. Pat. No. 4,029,084 (Purdue
Research Foundation), bacteriochlorophyll-a derivative useful in
photodynamic therapy in U.S. Pat. No. 5,173,504 (Health Research Inc),
purified hematoporphyrin dimers and trimers useful in photodynamic therapy
in U.S. Pat. No. 5,190,966 (Health Research Inc), drugs comprising
porphyrins in U.S. Pat. No. 5,028,621 (Health Research Inc), hemoporphyrin
derivatives and process of preparing in U.S. Pat. No. 4,866,168 (Health
Research Inc), method to destroy or impair target cells in U.S. Pat. No.
5,145,863 (Health Research Inc), method to diagnose the presence or
absence of tumor tissue in U.S. Pat. No. 5,015,463 (Health Research Inc),
photodynamic therapeutic technique in U.S. Pat. No. 4,957,481 (U.S.
Bioscience), apparatus for examining living tissue in U.S. Pat. No.
2,437,916 (Philip Morris and Company), transillumination method apparatus
for the diagnosis of breast tumors and other breast lesions by
normalization of an electronic image of the breast in U.S. Pat. No.
5,079,698 (Advanced Light Imaging Technologies), tricarbocyanine infrared
absorbing dyes in U.S. Pat. No. 2,895,955 (Eastman Kodak), optical imaging
system for neurosurgery in CA 2,048,697 (Univ. Techn. Int.), new porphyrin
derivatives and their metallic complexes as photosensitizer for PDT in
diagnosis and/or treatment of cancer in JP 323,597 (Hogyo,T), light
receiving system of heterodyne detection and image forming device for
light transmission image in EP 445,293 (Research Development Corp. of
Japan), light receiving system of heterodyne detection and image forming
device for light transmission image using light receiving system in WO
91/05239 (Research Development Corp. of Japan), storage-stable porphyrin
compositions and a method for their manufacture in U.S. Pat. No. 4,882,234
(Healux), method for optically measuring chemical analytes in WO 92/19957
(Univ. of Maryland at Baltimore), wavelength-specific cytotoxic agents in
U.S. Pat. No. 4,883,790 (Univ. of British Columbia),
hydro-monobenzo-porphyrin wavelength-specific cytotoxic agents in U.S.
Pat. No. 4,920,143 (Univ. of British Columbia), apparatus and method for
quantitative examination and high-resolution imaging of human tissue in EP
447,708 (Haidien Longxing Med Co), optical imaging system for neurosurgery
in U.S. application Ser. No. 7,565,454 (University Technologies Int.
Inc.), --characterization of specific drug receptors with fluorescent
ligands in WO 93/03382 (Pharmaceutical Discovery Corp),
4,7-dichlorofluorescein dyes as molecular probes in U.S. Pat. No.
5,188,934 (Applied Biosystems), high resolution breast imaging device
utilizing non-ionizing radiation of narrow spectral bandwith in U.S. Pat.
No. 4,649,275 (Nelson, R. et al.),
meso-tetraphenyl-porphyrin-Komplexverbindungen, Verfaren zu ihrer
Herstellung und Diese Enthaltends Pharmazeutische Mittel in EP 336,879
(Schering), 13,17-propionsaure und propionsaurederivat Substituerte
Porphyrin-Komplexverbindungen, Verfahren zu ihrer Herstellung und diese
Enthaltende Pharmazeutische Mittel in EP 355,041 (Schering),
photosensitizing agents in U.S. Pat. No. 5,093,349 (Health Research),
pyropheophorbides and their use in photodynamic therapy in U.S. Pat. No.
5,198,460 (Health Research), optical histochemical analysis, in vivo
detection and real-time guidance for ablation of abnormal tissues using
Raman spectroscopic detection system in WO 93/03672 (Redd, D.),
tetrabenztriazaporphyrin reagents and kits containing the same in U.S.
Pat. No. 5,135,717 (British Technology Group), system and method for
localization of functional activity in the human brain in U.S. Pat. No.
5,198,977 (Salb, J.). photodynamic activity of sapphyrins in U.S. Pat. No.
5,120,411 (Board of Regents, University of Texas), process for preparation
of expanded porphyrins in U.S. Pat. No. 5,152,509 (Board of Regents,
University of Texas), expanded porphyrins (Board of Regents, University of
Texas), infrared radiation imaging system and method in WO 88/01485
(Singer Imaging), imaging using scattered and diffused radiation in WO
91/07655 (Singer Imaging), diagnostic apparatus for intrinsic fluorescence
of malignant tumor in U.S. Pat. No. 4,957,114, indacene compounds and
methods for using the same in U.S. Pat. No. 5,189,029 (Bo-Dekk Ventures),
method of using 5,10,15,20-tetrakis (carboxy phenyl) porphine for
detecting cancers of the lung in U.S. Pat. No. 5,162,231 (Cole, D. A. et
al.), Verfahren zur Abbildung eines Gewebebereiches in DE 4327 798
(Siemens), chlorophyll and bacteriochlorophyll derivatives, their
preparation and pharmaceutical compositions comprising them in EPO 584 552
(Yeda Research and Development Company), wavelength-specific
photosensitive porphacyanine and expanded porphyrin-like compounds and
methods for preparation and use thereof in WO 94/10172 (Qudra Logic
Technologies), method and apparatus for improving the signal to noise
ratio of an image formed of an object hidden in or behind a semiopaque
random media in U.S. Pat. No. 5,140,463 (Yoo, K. M. et al.),
benzoporphyrin derivatives for photodynamic therapy in U.S. Pat. No.
5,214,036 (University of British Columbia), fluorescence diagnostics of
cancer using delta-amino levulinic acid in WO 93/13403 (Svanberg et al.),
Verfahren zum Diagnostizieren von mit fluoreszierenden Substansen
angereicherten, inbesondere tumorosen Gewebebereichen in DE 4136 769
(Humboldt Universitat), terpyridine derivatives in WO 90/00550 (Wallac).
All the light imaging dyes or contrast agents described in the
state-of-the-art have different properties, but all those agents have an
effect on the incident light, leading to either absorption and/or
fluorescence. However none of these contrast agents is used as a
particulate contrast agent.
SUMMARY OF THE INVENTION
We have now found that contrast enhancement may be achieved particularly
efficiently in light imaging methods by introducing particulate materials
as scattering contrast agents. For the sake of clarity, the word
"particle" is used to refer to any physiologically acceptable particulate
materials. Such particles may be solid (e.g. coated or uncoated
crystalline materials) or fluid (e.g. liquid particles in an emulsion) or
may be aggregates (e.g. fluid containing liposomes). Particulate material
with a particle size smaller than or similar to the incident light
wavelength are preferred.
Thus viewed from one aspect the invention provides the use of a
physiologically tolerable particulate material for the manufacture of a
particulate-contrast-agent containing contrast medium for use in in vivo
diagnostic light imaging.
Viewed from a further aspect the invention also provides a method of
generating an image of the human or non-human (preferably mammalian, avian
or reptilian) animal body by light imaging, characterised in that a
contrast effective amount of a physiologically tolerable particulate
contrast agent is administered to said body, and an image of at least part
of said body is generated. In such a method a contrast effective amount of
the particulate agent is administered, e.g. parenterally or into an
externally voiding body organ or duct, light emitted, transmitted or
scattered by the body is detected and an image is generated of at least
part of the body in which the contrast agent is present. Hybrid methods in
which light, either alone or in conjunction with other forms of radiation,
is administered to the body, and light, or some other form of radiation,
is detected. In particular, the other form of radiation may be ultrasound.
DETAILED DESCRIPTION OF THE INVENTION
The particles used according to the invention are preferably
water-insoluble or at least sufficiently poorly soluble as to retain their
desired particle size (e.g. 600-1300 nm) for at least 2 hours following
administration into the body under investigation.
The images generated may be spatial or temporal and mono- or
multi-dimensional.
In a further aspect of the invention, the imaging technique may be used to
determine a value for a parameter characteristic of the body or the part
of the body under study, e.g. blood flow rate. In this case however, the
parameter determination should be based on light detected from particles
studied through the skin or through an endoscopically or surgically
exposed surface.
Particularly preferably, the light imaging procedure used is selected from
confocal scanning laser microscopy (CSLM), optical coherence tomography
(OCT), laser doppler, laser speckle, and multi-photon microscopy
techniques (for a description of the latter see for example Denk, W. in
Photonics Spectra (1997) July 125-130, Denk, W. et al. in Science (1990)
April 248 73-76, Denk, W. et al. in J. Neurosci.Meth. (1994) 54:2:151-162,
Denk, W. et al. in Neuron (1997) January 18:351-357, Maiti, S. et al. in
Science (1997) January 275 530-532 and Denk, W. et al. in Proc. Natl.
Acad. (1995) August 92:18:8279-8282).
Confocal scanning laser microscopy (CSLM) is an imaging modality that
selectively detects a single point within a test object by focusing light
from a pinhole source onto that point. The light transmitting past or
reflecting from that point is refocused onto a second pinhole that filters
out light coming from any other site in the object except the focal point.
Raster scanning of the focus point through a plane passing through the
sample generates a full image of that plane of points. Moving the pinholes
and focusing apparatus back and forth from the sample selects out
different sample planes. In effect CSLM is a means for "optically"
sectioning a test sample. It pulls out images of individual sections of
the sample, but without the necessity that those sections be physically
separated from the rest of the sample.
Optical coherence tomography (OCT) accomplishes optical sectioning in a
related, but somewhat different manner. A collimated beam of light is
reflected from the sample, then is compared with a reference beam that has
travelled a precisely known distance. Only the light travelling exactly
the same distance to the sample and back as the distance the reference
beam travels from the source to the detector constructively interferes
with the reference beam and is detected. Thus the light from a single
plane within the sample is again selected. Varying the distance that the
reference beam travels before it is compared with the sampling beam
selects out different sample planes.
CSLM, OCT, laser doppler and laser speckle are discussed for example by:
Rajadhyaksha et al. in Laser Focus World, February 1997, pages 119 to 127;
Sabel et al. in Nature Medicine 3(2): 244-247 (1997); Tearney et al. in
SPIE 2389: 29-34 (1995); Bonner et al. in "Scattering techniques applied
to supramolecular and non-equilibrium systems", pages 685-701, Ed. Chen et
al., Plenum; Ruth in J. Microcirc: Clin Exp 9: 21-45 (1990); Pierard in
Dermatology 186: 4-5 (1993); and Bonner et al. in "Laser-doppler blood
flowmetry" pages 17 to 46, Ed. Shepherd et al., Kluwer, 1990.
CSLM and OCT may be used particularly effectively to study structures and
events occurring in the skin or within about a millimeter of an accessible
surface of the body under study, e.g. a surface exposed during surgical
operation or exposed endoscopically.
CSLM and OCT can be useful in optically guided tumor resection. For
example, either device attached to a colonoscope may facilitate
determination that no residual malignant tissue remains after removal of a
cancerous colon polyp. Additional applications include, but are not
limited to, diagnosis and treatment of disease in the rest of the
digestive tract, surgical treatment of ulcerative colitis, and diagnosis
and treatment of endometriosis.
Dynamically, CSLM and OCT can be used to follow the movement of blood cells
through the capillaries of the skin and other vascularized tissue lying
within about a millimeter of an exposed surface. Potentially they can also
be used in conjunction with laser Doppler or speckle inferferometry for
the measure of blood flow.
Laser Doppler and speckle interferometry are related, each relying upon the
fact that the intensity of light detected after a beam of laser light that
interacts with a collection of moving particles changes with time.
Mathematical analysis of the changes provides a basis for calculating the
rate at which the particles are moving.
The perfusion of tissue that is exposed by surgery is one important
indicator of the health of that tissue. Blood flow within the skin of the
breast may be an indicator of internal disease. Blood flow in the skin can
be detected by laser Doppler blood-flow measurement or laser speckle
interferometry, either by itself or in conjunction with CSLM or OCT.
According to the present invention, synthetic particles, capable of
scattering light of the wavelength used for the imaging procedure, may be
administered as contrast agents in an in vivo light imaging procedure.
Typically such scattering particles will be administered in suspension in
a physiologically tolerable fluid (e.g. water for injections,
physiological saline, Ringer's solution etc.) into the vasculature or
musculature or into the tissue or organ of interest.
A preferred contrast agent for intraoperative CSLM or OCT will have the
following properties: it will consist of stabilized particles in an
aqueous or buffered liquid medium. The particle size will preferably be
around 600 to 1300 nm, more preferably 700 to 1100 nm (i.e. roughly equal
to the wavelength of the light source). The refractive index of the
particles will preferably differ from that of body fluids, such as blood
and lymph, by at least 0.01. Optionally the particles may have fluorescent
dyes attached to their surfaces or contained within them or the particles
themselves may be composed of fluorescent dyes. Optionally the particles
may have suitable surface modifying agents, such as poly(ethylene glycol),
to slow their uptake by macrophages in the body and to prolong their blood
circulation lifetimes.
The particles may be of a material which is transparent or translucent or
more preferably opaque to light of the wavelength of the light source.
Particularly preferably, the particles are substantially monodisperse
polymer particles (with a coefficient of variation of the particle size
(i.e. 100.times.standard deviation.div.mean particle size by volume of the
major mode of the detectable particles) as measured by a Coulter LS 130
particle size analyzer of less than 10%, preferably less than 5%). Such
particles may be prepared by the SINTEF technique disclosed in U.S. Pat.
No. 4,336,173 and U.S. Pat. No. 4,459,378. Such polymer particles may be
simple scatterers or may be modified to carry a chromophore (or
fluorophore), preferably having characteristic absorption and/or emission
maxima in the 600 to 1300 nm range. Furthermore they may be modified to
include or carry a targetting vector, e.g. a species serving to cause the
particles to accumulate at a desired target site, for example
superparamagnetic crystals which allow the particle to be accumulated at a
target site by application of an external magnetic field, or a drug,
antibody, antibody fragment or peptide (e.g. an oligopeptide or
polypeptide) which has a binding affinity for sites within the target
zone, e.g. cell surface receptors.
The particulate contrast agent can be applied through simple topical
application or other pharmaceutically acceptable routes. For
dermatological applications, the contrast agents may be modified to be
delivered through transdermal patches or by iontophoretesis. Iontophoretic
delivery is preferred, as one can control the amount of the agent that is
delivered.
For intraoperative uses the contrast agent can be injected into the
vasculature or into the lesion to be removed prior to or during the
surgery. For detection of lymph nodes it can be injected into a lymph duct
draining into the surgical area. Alternatively it may be applied during
surgery as a topical ointment, a liquid, or a spray. For measurement of
blood flow the agent can be injected intravascularly prior to the
measurement.
As indicated above, the particulate agents used according to the invention
may comprise a chromophore or fluorophore, i.e. may absorb or emit light
in the wavelength range detected in the imaging procedure or alternatively
may rely primarily upon light scattering effects. In the latter case, one
may simply use physiologically tolerable non photo-labelled particles,
e.g. particles of an inert organic or inorganic material, e.g. an
insoluble triiodophenyl compound or titanium dioxide, which appears white
or colourless to the eye. Where the particles comprise a fluorophore or
chromophore, i.e. are photo-labelled, this may be in a material carried by
(e.g. bound to, coated on, or contained or deposited within) a particulate
carrier (e.g. a solid particulate or a liposome). Alternatively the
carrier itself may have chromophoric or fluorophoric properties. While the
photolabel may be a black photolabel (i.e. one which absorbs across the
visible spectrum and thus appears black to the eye) non-black photolabels
are preferred.
Scattering contrast agents (and absorbing contrast agents for that matter)
can have several mechanisms in image enhancement for light imaging
applications. The first mechanism is a direct image enhancing role similar
to the effect that x-ray contrast media have in x-ray imaging. In direct
image enhancement, the contrast medium contributes directly to an
improvement in image contrast by affecting the signal intensity emanating
from the tissue containing the contrast medium. In light imaging,
scattering (and absorbing) agents localized in a tissue can attenuate
light differently than the surrounding tissue, leading to contrast
enhancement.
For near surface methods such as confocal microscopy and optical coherence
tomography, scattering agents generate contrast primarily by serving as
reflection centres that selectively direct the incident light to the
detector. When scattering sites are trapped in a moving fluid, such as
blood, the extent of the scattering sites' movement can be used as a
measure of the fluid's flow rate.
The "speckle" phenomenon results from the interaction of coherent radiation
(such as that from a laser) with scattering sites. When the scattering
sites move, the speckle pattern changes with time, and the rate of change
of the speckle pattern can be used to determine the rate of movement of
the scattering sites. If the movement of the scattering sites is
non-random, for example when they are entrained in a moving fluid, the
rate of fluid flow can be determined by the changes in the speckle pattern
over time.
A second mechanism by which a scattering (or absorbing) agent could be used
is as a noise rejection agent. The contrast agent in this case is not
directly imaged as described above, but functions to displace a noise
signal from an imaging signal so that the desired signal is more readily
detected. Noise in light imaging applications results from multiple
scattering and results in a degradation of image quality. The origin of
this noise is as follows:
As previously mentioned, light propagating through a random medium such as
tissue undergoes multiple scattering. This scattering splits the incident
light into three components, the ballistic, quasi-ballistic, and
incoherent (highly scattered) components. The ballistic and
quasi-ballistic signals propagate through tissue in the forward direction
and carry the object information. The incoherent component constitutes
noise because the light has undergone random scattering in all directions
and information about the object is lost. When the intensity of the
ballistic and quasi-ballistic signals are reduced below the intensity of
the multiply scattered noise, the object becomes invisible. This multiple
scattering noise can be partially removed by a spatial filter that rejects
light scattered away from the collinear direction of the incident light.
However, a substantial portion of noise emerges from the object after
multiple scattering events by rejoining the original ballistic signal.
This multiply scattered light can not be removed by spatial filtering due
to its collinear path with the desired ballistic signal.
Scattering (and absorbing) agents can aid in the removal of unwanted noise
component from the desired ballistic and quasi-ballistic signals. This is
based on the fact that multiply scattered light undergoes a random walk in
tissue and thus travels over a longer path length than the ballistic
signal. The distance the ballistic and quasi-ballistic signals traverses
is essentially the thickness of the tissue (or body part) being imaged.
Scattered light traveling a longer distance has a greater probability of
being attenuated. Current technology uses a time-gate (temporal filter) to
reject the scattered signal (longer traveling=longer residence time in
tissue) from the ballistic and quasi-ballistic components.
The introduction of a small isotropic scattering agent greatly increases
the residence time of the highly scattered signal component while having a
lesser effect on the ballistic and quasi-ballistic components. This
effectively provides a longer separation between the ballistic and
quasi-ballistic signals and the highly scattered component, providing
improved rejection of the scattered (noise) component and better image
quality.
Very little is disclosed in prior art regarding particulate
scattering-based contrast agents. To our knowledge the only prior art with
regard to particulate scattering-based contrast agents is U.S. Pat. No.
5,140,463 (Yoo, K. M. et al.) which discloses a method and apparatus for
improving the signal to noise ratio of an image formed of an object hidden
in or behind a semi-opaque medium. The patent in general terms suggests to
make the random medium less random (so that there will be less scattered
light) and it is also suggested to increase the time separation between
ballistic and quasi-ballistic light and the highly scattered light. One of
many ways to obtain this will, according to the patent, be to introduce
small scatterers into the random medium. There are no further suggestions
regarding these small scatterers and no suggestion of in vivo use.
Particulate materials in the form of liposomes have been suggested;
liposome or LDL-administered Zn(II)-phthalocyanine has been suggested as
photodynamic agent for tumors by Reddi, E. et al. in Lasers in Medical
Science 5 (1990) 339, parenterally administered zinc phtalocyanine
compositions in form of liposome dispersion containing synthetic
phopholipid in EP 451 103 (CIBA Geigy) and liposome compositions
containing benzoporphyrin derivatives used in photodynamic cancer therapy
or an antiviral agents in CA 2,047,969 (Liposome Company). These
particulate materials have been suggested as therapeutic agents and have
nothing to do with scattering light imaging contrast agents.
In one embodiment of the invention the contrast medium for imaging
modalities based on light will comprise physiologically tolerable gas
containing particles. Preferred are e.g. biodegradable gas-containing
polymer particles, gas-containing liposomes or aerogel particles.
This embodiment of the invention includes, for example, the use in light
imaging of particles with gas filled voids (U.S. Pat. No. 4,442,843),
galactose particles with gas (U.S. Pat. No. 4,681,119), microparticles for
generation of microbubbles (U.S. Pat. No. 4,657,756 and DE 3313947),
protein microbubbles (EP 224934), clay particles containing gas (U.S. Pat.
No. 5,179,955), solid surfactant microparticles and gas bubbles (DE
3313946), gas-containing microparticles of amylose or polymer (EP 327490),
gas-containing polymer particles (EP 458079), aerogel particles (U.S. Pat.
No. 5,086,085), biodegradable polyaldehyde microparticles (EP 441468), gas
associated with liposomes (WO 9115244), gas-containing liposomes (WO
9222247), and other gas containing particles (WO 9317718, EP 0398935, EP
0458745, WO 9218164, EP 0554213, WO 9503835, DE 3834705, WO 9313809, WO
9112823, EP 586875, WO 9406477, DE 4219723, EP 554213, WO 9313808, WO
9313802, DE 4219724, WO 9217212, WO 9217213, WO 9300930, U.S. Pat. No.
5,196,183, WO 9300933, WO 9409703, WO 9409829, EP 535387, WO 9302712, WO
9401140). The surface or coating of the particle can be any
physiologically acceptable material and the gas can be any acceptable gas
or gas mixture. Specially preferred gases are the gases used in ultrasound
contrast agents like for example air, nitrogen, lower alkanes and lower
fluoro or perfluoro alkanes (e.g. containing up to 7, especially 4, 5 or 6
carbons).
Where gas microbubbles (with or without a liposomal encapsulating membrane)
are used according to the invention, advantage may be taken of the known
ability of relatively high intensity bursts of ultrasound to destroy such
microbubbles. Thus by comparing the detected light signal (or image)
before and after ultrasound exposure mapping the distribution of the
contrast agent may be facilitated.
In another embodiment of the invention the contrast medium for imaging
modalities based on light will comprise physiologically tolerable
particles of lipid materials, e.g. emulsions, especially aqueous
emulsions. Preferred are halogen comprising lipid materials. This
embodiment of the invention includes, for example, the use in light
imaging of fat emulsions (JP 5186372), emulsions of fluorocarbons (JP
2196730, JP 59067229, JP 90035727, JP 92042370, WO 930798, WO 910010, EP
415263, WO 8910118, U.S. Pat. No. 5,077,036, EP 307087, DE 4127442, U.S.
Pat. No. 5,114,703), emulsions of brominated perfluorocarbons (JP
60166626, JP 92061854, JP 5904630, JP 93001245, EP 231070),
perfluorochloro emulsions (WO 9311868) or other emulsions (EP 321429).
In yet another embodiment of the invention the contrast medium for imaging
modalities based on light will comprise physiologically tolerable
liposomes. Preferred groups of liposomes are phospholipid liposomes and
multilamelar liposomes. This embodiment of the invention includes, for
example, the use in light imaging of phospholipid liposomes containing
cholesterol derivatives (U.S. Pat. No. 4,544,545); liposomes associated
with compounds containing aldehydes (U.S. Pat. No. 4,590,060); lipid
matrix carriers (U.S. Pat. No. 4,610,868); liposomes containing
triiodobenzoic acid derivatives of the type also suitable for X-ray
examination of liver and spleen (DE-2935195); X-ray contrast liposomes of
the type also suitable for lymphography (U.S. Pat. No. 4,192,859);
receptor-targeted liposomes (WO-8707150); immunoactive liposomes
(EP-307175); liposomes containing antibody specific for antitumor antibody
(U.S. Pat. No. 4,865,835); liposomes containing oxidants able to restore
MRI contrast agents (spin labels) which have been reduced (U.S. Pat. No.
4,863,717); liposomes containing macromolecular bound paramagnetic ions of
the type also suitable for MRI (GB-2193095); phospholipid liposomes of the
type also suitable for ultrasound imaging containing sodium bicarbonate or
aminomalonate as gas precursor (U.S. Pat. No. 4,900,540); stable
plurilamellar vesicles (U.S. Pat. No. 4,522,803); oil-filled paucilamellar
liposomes containing non-ionic surfactant as lipid (U.S. Pat. No.
4,911,928); liposomal phospholipid polymers containing ligands for
reversible binding with oxygen (U.S. Pat. No. 4,675,310); large
unilamellar vesicle liposomes containing non-ionic surfactant (U.S. Pat.
No. 4,853,228); aerosol formulations containing liposomes (U.S. Pat. No.
4,938,947 and U.S. Pat. No. 5,017,359); liposomes containing amphipathic
compounds (EP-361894); liposomes produced by adding an aqueous phase to an
organic lipid solution followed by evaporating the solvent and then adding
aqueous lipid phase to the concentrate (FR-2561101); stable monophasic
lipid vesicles of the type also useful for encapsulation of bioactive
agents at high concentrations (WO-8500751); homogeneous liposome
preparations (U.S. Pat. No. 4,873,035); stabilized liposome compounds
comprising suspensions in liquefiable gel (U.S. Pat. No. 5,008,109);
lipospheres (solid hydrophilic cores coated with phospholipid) of the type
also suitable for controlled extended release of active compounds
(WO-9107171); liposomes sequestered in gel (U.S. Pat. No. 4,708,861);
metal chelates bound to liposomes, also suitable for use as MR contrast
agents (WO-9114178); lipid complexes of X-ray contrast agents
(WO-8911272); liposomes which can capture high solute to lipid ratios
(WO-9110422); liposomes containing covalently bound PEG moieties on
external surface to improve serum half-life (WO-9004384); contrast agents
comprising liposomes of specified diameter encapsulating paramagnetic
and/or superparamagnetic agents (WO-9004943); liposomes of the type also
suitable for delivering imaging agents to tumours consisting of small
liposomes prepared from pure phopholipids (EP-179444); encapsulated X-ray
contrast agents such as iopromide in liposomes (U.S. Pat. No. 5,110,475);
non-phospholipid liposome compositions (U.S. Pat. No. 5,043,165 and U.S.
Pat. No. 5,049,389); hepatocyte-directed vesicle delivery systems (U.S.
Pat. No. 4,603,044); gas-filled liposomes of the type also suitable as
ultrasound contrast agents for imaging organs (U.S. Pat. No. 5,088,499);
injectable microbubble suspensions stabilized by liposomes (WO-9115244);
paramagnetic chelates bound to liposomes (U.S. Pat. No. 5,135,737);
liposome compositions of the type also suitable for localising compounds
in solid tumors (WO-9105546); injectable X-ray opacifying liposome
compositions (WO-8809165); encapsulated iron chelates in liposomes
(EP-494616); liposomes linked to targeting molecules through disulphide
bonds (WO-9007924); and compositions consisting of non-radioactive
crystalline X-ray contrast agents and polymeric surface modifiers with
reduced particle size (EP-498482).
Water soluble compounds which, in simple aqueous solution are not
apparently significant light scatterers or absorbers, may become efficient
scatterers on incorporation within liposomes. Thus iodixanol (and other
soluble iodinated X-ray contrast agents that are commercially available)
provides a clear solution on dissolution in water. However when iodixanol
is encapsulated in liposomes the resulting particulate product is
off-white indicating a significant light scattering capability.
Besides using liposomes as carriers for light imaging contrast agents, it
is possible to use simple micelles, formed for example from surfactant
molecules, such as sodium dodecyl sulphate, cetyltrimethylammonium
halides, pluronics, tetronics etc., as carriers for photolabels which are
moderately or substantially water insoluble but are solubilised by the
amphiphilic micelle forming agent, e.g. photolabels such as indocyanine
green. Similarly peptides such as PEG modified polyaspartic acid (see Kwon
et al. Pharm. Res. 10: 970 (1993)) which spontaneously aggregate into
polymeric micelles may be used to carry such photolabels. Likewise
photolabel carrier aggregate particles can be produced by treatment of
polycyclic aromatic hydrocarbons with anionic surfactants (e.g. sodium
dodecyl sulphate or sulphated pluronic F108) and subsequent addition of
heavy metal ions (e.g. thorium or silver). Such heavy metal treatment
gives rise to micelles exhibiting phosphorescent behaviour and these can
be used in the present invention without incorporation of a photolabel,
especially using a pulsed light source and gated detection of the
temporally delayed phosphorescent light.
In a still further embodiment of the invention the contrast medium for
imaging modalities based on light will comprise physiologically tolerable
particles containing iodine. These particles may for example be particles
of a substantially water insoluble solid or liquid iodine-containing
compound, e.g. an inorganic or organic compound, in the latter case
preferably a triiodophenyl group containing compound, or alternatively
they may be aggregate particles (such as liposomes) in which at least one
of the components is an iodinated compound. In this case the iodinated
compound may be a membrane forming compound or may be encapsulated by the
membrane. For example, the use of emulsified iodinated oils (U.S. Pat. No.
4,404,182), particulate X-ray contrast agents (JP 67025412, SU 227529, DE
1283439, U.S. Pat. No. 3,368,944, AU 9210145, EP 498482, DE 4111939, U.S.
Pat. No. 5,318,767), iodinated esters (WO 9007491, EP 300828, EP 543454,
BE 8161143) and iodinated lipids (EP 294534) are included in this
embodiment of the invention.
In a yet still further embodiment of the invention the contrast medium for
imaging modalities based on light will comprise physiologically tolerable
magnetic particles. The term "magnetic particle" as used here means any
particle displaying ferromagnetic, ferrimagnetic or superparamagnetic
properties and preferred are composite particles comprising magnetic
particles and a physiologically tolerable polymer matrix or coating
material, e.g. a carbohydrate and/or a blood residue prolonging polymer
such as a polyalkyleneoxide (e.g. PEG) as described for example by
Pilgrimm or Illum in U.S. Pat. No. 5,160,725 and U.S. Pat. No. 4,904,479
e.g. biodegradable matrix/polymer particles containing magnetic materials.
This embodiment of the invention includes, for example, the use in light
imaging of magnetic liquid (SU 1187221), ferrite particles coated with a
negatively charged colloid (DE 2065532), ferrite particles (U.S. Pat. No.
3,832,457), liquid microspheres containing magnetically responsive
substance (EP 42249), magnetic particles with metal oxide core coated with
silane (EP 125995), magnetic particles based on protein matrix (DE
3444939), magnetic vesicles (JP 60255728), magnetic particles (SU 106121),
magnetic particles embedded in inert carrier (JP 62167730), ferromagnetic
particles loaded with specific antibodies (DE 3744518), superparamagnetic
particles coated with biologically acceptable carbohydrate polymers (WO
8903675), polymerized lipid vesicles containing magnetic material (U.S.
Pat. No. 4,652,257), superparamagnetic materials in biodegradable matrices
(U.S. Pat. No. 4,849,210), biodegradable matrix particles containing
paramagnetic or ferromagnetic materials (U.S. Pat. No. 4,675,173),
ferromagnetic particles with substances for binding affinity for tissue
(WO 8601112), ferrite particles (JP 47016625, JP 47016624), ferromagnetic
particles (NL 6805260), magnetic polymer particles (WO 7800005, JP
62204501, JP 94016444, WO 870263), barium ferrite particles (WO 8805337),
magnetic iron oxide particles (U.S. Pat. No. 4,452,773), amino acid
polymer containing magnetic particles (U.S. Pat. No. 4,247,406), complexed
double metal oxide particles (EP 186616), magnetic particles (GB 2237198),
encapsulated superparamagnetic particles (WO 8911154), biodegradable
magnetic particles (WO 8911873), magnetic particles covalently bond to
proteins (EP 332022), magnetic particles with carbohydrate matrix (WO
8301768), magnetic particles with silicon matrix (EP 321322), polymer
coated magnetic particles (WO 9015666), polymer-protected collodial metal
dispersion (EP 252254), biodegradable superparamagnetic particles (WO
8800060), coated magnetic particles (WO 9102811), ferrofluid (DE 4130268),
organometallic coated magnetic particles (WO 9326019) and other magnetic
particles (EP 125995, EP 284549, U.S. Pat. No. 5,160,726, EP 516252, WO
9212735, WO 9105807, WO 9112025, WO 922586, U.S. Pat. No. 5,262,176, WO
9001295, WO 8504330, WO 9403501, WO 9101147, EP 409351, WO 9001899, EP
600529, WO 9404197).
The particulate contrast agent used according to the invention may, as
mentioned above, be non-photo-labelled or photolabelled. In the latter
case this means that the particle either is an effective photoabsorber at
the wavelength of the incident light (i.e. carries a chromophore) or is a
fluorescent material absorbing light of the incident wavelength and
emitting light at a different wavelength (i.e. carries a fluorophore).
Examples of suitable fluorophores include fluorescein and fluorescein
derivatives and analogues, indocyanine green, rhodamine,
triphenylmethines, polymethines, cyanines, phalocyanines, naphthocyanines,
merocyanines, lanthanide complexes (e.g. as in U.S. Pat. No. 4,859,777) or
cryptates, etc. including in particular fluorophores having an emission
maximum at a wavelength above 600 nm (e.g. fluorophores as described in
WO-A-92/08722). Other labels include fullerenes, oxatellurazoles (e.g. as
described in U.S. Pat. No. 4,599,410), LaJolla blue, porphyrins and
porphyrin analogues (e.g. verdins, purpurins, rhodins, perphycenes,
texaphyrins, sapphyrins, rubyrins, benzoporphyrins, photofrin,
metalloporphyrins, etc.) and natural chromophores/fluorophores such as
chlorophyll, carotenoids, flavonoids, bilins, phytochrome, phycobilins,
phycoerythrin, phycocyanins, retinoic acid and analogues such as retinoins
and retinates.
In general, photolabels which contain chromophores should exhibit a large
molar absorptivity, e.g. >10.sup.5 cm.sup.-1 M.sup.-1 and an absorption
maximum in the optical window 600 to 1300 nm. Particulates for use as
noise rejection agents by virtue of their absorption properties should
similarly preferably have molar absorptivities in excess of 10.sup.5
cm.sup.-1 M.sup.-1 and an absorption maximum in the range 600 to 1300
nm.sup.-1. For fluorescent particles, the quantum yield for fluorescence
is one of the most important characteristics. This should be as high as
possible. However the molar absorptivity should also desirably be above
10.sup.5 cm.sup.-1 M.sup.-1 for the fluorophore and the absorption maximum
should desirably be in the range 600 to 1300 nm for diffuse reflectance
studies or 400 to 1300 nm for surface studies.
These photo-labelled materials may be used as such if substantially
water-insoluble and physiologically tolerable, e.g. as solid or liquid
particles, or alternatively may be conjugated to or entrapped within a
particulate carrier (e.g. an inorganic or organic particle or a liposome).
Particularly preferred in this are conjugates of formula I
I.sub.3 Ph-L-C* (I)
where I.sub.3 Ph is a triiodophenyl moiety, L is a linker moiety and C* is
a chromophore or fluorophore (e.g. as described above). Such compounds
form a further aspect of the invention.
The I.sub.3 Ph moiety is preferably a 2, 4, 6 triiodo moiety having
carboxyl or amine moieties (or substituted such moieties, e.g.
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
alkoxycarbonylalkoxycarbonyl, or alkylcarbonylamino groups where the alkyl
or alkylene moieties are optionally hydroxy substituted and preferably
contain up to 20, particularly 1 to 6, especially 1 to 3 carbons) at the 3
and 5 positions. The linker group L may be any group capable of linking
the group C* to the I.sub.3 Ph moiety, e.g. an amide, amine, NHSO.sub.2 or
carboxyl group or a thio analog thereof; or a C.sub.1-20. alkylene chain
terminated by such groups and optionally with one or more methylene groups
replaced by thia or oxa and optionally substituted for example by thio,
oxo, hydroxy or alkyl moieties. Examples of group L include --NHSO.sub.2
-- and --CO.sub.2 (CH.sub.2).sub.2 O--CS--NH--.
Such compounds may be prepared by conjugating a chromophoric or
fluorophoric molecule to a triiodophenyl compound of the type proposed as
X-ray contrast agents by Nycomed, Sterling Winthrop, or Bracco in their
numerous patent publications (by way of example U.S. Pat. No. 5,264,610,
U.S. Pat. No. 5,328,404, U.S. Pat. No. 5,318,767 and U.S. Pat. No.
5,145,684).
In one particular embodiment of the invention, non-photolabelled particles,
e.g. solid particles of a polymer or an iodinated X-ray contrast agent,
are provided with a coating or shell of a photolabel, e.g. a fluorescent
agent, for example by chemically or physiochemically binding the
photolabel to the particles (e.g. by using oppositely charged photolabel
and particles). The resulting coated particles, preferably of nano
particle size (e.g. 5 to 800 nm, especially 10 to 500 nm) if labelled with
a fluorophore would allow light energy trapped by the core to be
transferred to the luminescing surface and so enhance light emission by
the fluorophore. Compositions containing such particles form a further
aspect of the invention.
Alternatively the photo-label may be entrapped within a solid polymer
matrix, e.g. by co-precipitation of polymer and photolabel or by
precipitation of photo-label within the pores of a porous inorganic or
organic matrix.
Suitable organic polymer matrices for use as carriers or cores for
photolabels are substantially water insoluble physiologically tolerable
polymers, e.g. polystyrene latex, polylactide coglycolide,
polyhydroxybutyrate co-valerate etc.
Other physiologically acceptable particles may be used in contrast media
for imaging methods based on light in accordance with of the present
invention. Preferred groups of materials are e.g. biodegradable polymer
particles, polymer or copolymer particles and particles containing
paramagnetic materials. The particles can for example be crosslinked
gelatin particles (JP 60222046), particles coated with hydrophilic
substances (JP 48019720), brominated perfluorocarbon emulsions (JP
58110522), perfluorocarbon emulsions (JP 63060943), particles and
emulsions for oral use (DE 3246386), polymer particles (WO 8601524, DE
3448010), lipid vesicles (EP 28917), metal oxide particles (JP 1274768),
metal transferrin dextran particles (U.S. Pat. No. 4,735,796),
monodisperse magnetic polymer particles (WO 8303920), polymer particles
(DE 2751867), microparticles containing paramagnetic metal compounds (U.S.
Pat. No. 4,615,879), porous particles containing paramagnetic materials
(WO 8911874), hydrophilic polymer particles (CA 1109792), water-swellable
polymer particles (DE 2510221), polymer particles (WO 8502772), metal
loaded molecular sieves (WO 9308846), barium sulphate particles (SU
227529), metal particles (DE 2142442), crosslinked polysaccharide
particles (NL 7506757), biodegradable polymer particles (BE 869107),
niobium particles (SU 574205), biodegradable polymer particles (EP
245820), amphiphilic block copolymers (EP 166596), uniform size particles
(PT 80494), coloured particles (WO 9108776), polymer particles (U.S. Pat.
No. 5,041,310, WO 9403269, WO 9318070, EP 520888, DE 4232755), porous
polymer particles (WO 9104732), polysaccharide particles (EP 184899),
lipid emulsions (SU 1641280), carbohydrate particles (WO 8400294),
polycyanoacrylate particles (EP 64967), paramagnetic particles (EP
275215), polymer nanoparticles (EP 240424), nanoparticles (EP 27596, EP
499299), nanocapsules (EP 274961), inorganic particles (EP 500023, U.S.
Pat. No. 5,147,631, WO 9116079), polymer particles ((EP 514790), apatite
particles (WO 9307905), particulate micro-clusters (EP 546 939), gel
particles (WO 9310440), hydrophilic colloids (DE 2515426), particulate
polyelectrolyte complex (EP 454044), copolymer particles (EP 552802),
paramagnetic polymer particles (WO 9222201), hydrophilic poly-glutamate
microcapsules (WO 9402106) and other particles (WO 9402122, U.S. Pat. No.
4,997,454, WO 9407417, EP 28552, WO 8603676, WO 8807870, DE 373809, U.S.
Pat. No. 5,107,842, EP 502814).
In general, where the particulate agent is intended for parenteral
administration (e.g. into the vasculature), it may be desirable to prolong
the blood residence time for the particles by attaching to these a blood
residence time prolonging polymer as described for example by Pilgrimm in
U.S. Pat. No. 5,160,725 or Illum in U.S. Pat. No. 4,904,479. In this way
imaging of the vascular system may be facilitated by delaying the uptake
of the particle by the reticuloendothelial system. In the case of
liposomal particles, the blood residence prolonging polymer may be bound
to preformed liposomes or, conjugated to liposomal membrane forming
molecules, may be used as an amphiphilic membrane forming component so
resulting in liposomes carrying the hydrophilic blood residence polymer
component on their surfaces. Alternatively or additionally the particles
may be conjugated to a biotargetting moiety (e.g. as described in
WO-A-94/21240) so as to cause the particles to distribute preferentially
to a desired tissue or organ, e.g. to tumor tissue.
The particle size utilized according to the invention will depend upon
whether particle administration is parenteral or into an externally
voiding body cavity and on whether or not the particles are
photo-labelled. In general particle sizes will be in the range 5 to 10000
nm, especially 15 to 1500 nm, particularly 50 to 400 nm and for particles
which are being used for their scattering effect particle size will
preferably be in the range 1/15.lambda. to 2.lambda., or more preferably
1/10.lambda. to .lambda., especially .lambda./4.PI. to .lambda./.PI., more
especially about .lambda./2.PI. (where .lambda. is the wavelength of the
incident light in the imaging technique). By selecting a particle size
which scatters effectively at wavelengths above the absorption maxima for
blood, e.g. in the range 600 to 1000 nm, and by illuminating at a
wavelength in that range, the contrast efficacy of non-photolabelled
particles may be enhanced.
For administration to human or animal subjects, the particles may
conveniently be formulated together with conventional pharmaceutical or
veterinary carriers or excipients. The contrast media used according to
the invention may conveniently contain pharmaceutical or veterinary
formulation aids, for example stabilizers, antioxidants, osmolality
adjusting agents, buffers, pH adjusting agents, colorants, flavours,
viscosity adjusting agents and the like. They may be in forms suitable for
parenteral or enteral administration, for example, injection or infusion
or administration directly into a body cavity having an external voidance
duct, for example the gastrointestinal tract, the bladder and the uterus.
Thus the media of the invention may be in conventional pharmaceutical
administration forms such as tablets, coated tablets, capsules, powders,
solutions, suspensions, dispersions, syrups, suppositories, emulsions,
liposomes, etc; solutions, suspensions and dispersions in physiologically
acceptable carrier media, e.g. water for injections, will however
generally be preferred. Where the medium is formulated for parenteral
administration, the carrier medium incorporating the particles is
preferably isotonic or somewhat hypertonic.
The contrast agents can be used for light imaging in vivo, in particular of
organs or ducts having external voidance (e.g. GI tract, uterus, bladder,
etc.), of the vasculature, of phagocytosing organs (e.g. liver, spleen,
lymph nodes, etc.) or of tumors. The imaging technique may involve
endoscopic procedures, e.g. inserting light emitter and detector into the
abdominal cavity, the GI tract etc. and detecting transmitted, scattered
or reflected light, e.g. from an organ or duct surface. Where appropriate
monochromatic incident light may be utilized with detection being of
temporally delayed light emission (e.g. using pulsed light gated
detection) or of light of wavelengths different from that of the incident
light (e.g. at the emission maximum of a fluorophore in the contrast
agent). Similarly images may be temporal images of a selected target
demonstrating build up or passage of contrast agent at the target site.
The light used may be monochromatic or polychromatic and continuous or
pulsed; however monochromatic light will generally be preferred, e.g.
laser light. The light may be ultraviolet to near infra-red, e.g. 100 to
1300 nm wavelength however wavelengths above 300 nm and especially 600 to
1000 nm are preferred.
The contrast media of the invention should generally have a particle
concentration of 1 10.sup.6 g/ml to 50 10.sup.-3 g/ml, preferably 5
10.sup.-6 g/ml to 10 10.sup.-3 g/ml. Dosages of from 1 10.sup.-7 g/kg to 5
10.sup.-1 g/kg, preferably 1 10.sup.-6 g/kg to 5 10.sup.-2 g/kg will
generally be sufficient to provide adequate contrast although dosages of 1
10.sup.-4 g/kg to 1 10.sup.-2 g/kg will normally be preferred.
The various publications referred to herein are hereby incorporated by
reference.
The invention is further illustrated by the following non-limiting
Examples. Unless otherwise stated percentages and ratios are by weight.
EXAMPLE 1
Iodixanol Containing Liposomes
Liposomes of average diameter 300 to 600 nm are prepared by a modification
of the "Thin film hydration method" described by A. D. Bangham et al.
"Methods in Membrane Biology (E. D. Korn, ed), Plenum Press, N.Y., pp 1-68
(1974). The maximum batch size produced by the process is 2.0 L. The
hydrogenated phosphatidylcholine (10 g H-PC) and hydrogenated phosphatidyl
serine (1 g H-PS) are dissolved in chloroform/methanol/water (4:1:0.025,
volume ratios) by shaking in a water bath at 70.degree. C. The solvents
are removed by rotary evaporation until a dry mixture of the PLs appear.
The phospholipid mixture is added to an aqueous, isotonic solution of
iodixanol and tonicity agent at a temperature of 60-700C., and the mixture
is homogenised with a homomixer, (6000 rpm for 10 minutes at a temperature
of 65-70.degree. C.). The liposomes formed are extruded once through three
polycarbonate filters. 5.0 mL of the liposome suspension are filled in 20
mL glass bottles, closed with grey rubber stoppers and sealed with
aluminium capsules. The liposomes are sterilised by autoclaving (at
121.degree. C. for 20 minutes).
EXAMPLE 2
______________________________________
Fat emulsion
______________________________________
An oil-in-water emulsion is prepared from
soybean oil 10 g
safflower oil 10 g
egg phosphatides
1.2 g
glycerin 2.5 g
water to osmolarity of 258 mOsm/L and pH of 8.3 to
______________________________________
9.0
(Such an emulsion is available commercially under the trade name Liposyn II
from Abbott Laboratories, Chicago, Ill., U.S.A). This can be diluted with
physiological saline to the desired concentration.
EXAMPLE 3
A. Solid Microparticles
A gas-filled (e.g. air filled) microbubble suspension, with particle size 1
to 12 .mu.m may be prepared with oleic acid and human serum albumin as the
microbubble shell material.
A 216 ml sample of a 0.5% aqueous solution of sodium oleate was titrated
with 0.1 N HCl so that the final pH was in the range 3.9-4.0. The solution
had become very turbid due to the formation of an oleic acid suspension.
The particle size as measured by optical microscopy was in the 0.1 micron
range.
The suspension was pressurized to increase the solubility of the gas in the
oleic acid suspension. The suspension was placed in a 500 ml stirred
autoclave (Zipperclave manufactured by Autoclave Engineers, Inc.) fitted
with a 6 blade turbine-type impeller (from Dispersimax). The vessel was
sealed and charged to 1000 psig air (typical pressure ranges were 900-1100
psig). The suspension was agitated at 1000 rpm (agitation ranged from
750-1500 rpm) for one hour at room temperature (23-25.degree. C.).
Typically the temperature rose 2-3.degree. C. during the run. Agitation
was stopped, the vessel vented and the suspension was held for 30 minutes
before use. The particle size as measured by optical microscopy was in the
0.1 micron range.
2 g of a 25% aqueous solution of human serum albumin (HSA) was added to 28
g of water and 20 g of the emulsion described above. The turbid solution
was heated to 65.degree. C. while oxygen gas was bubbled in. The solution
was then stirred using an Omni-Stirrer (homogenizer) for 5 minutes at the
mid-range setting. The foamy mixture was poured into a separatory funnel
and left to stand for 30 minutes. The liquid was removed from the bottom
and 10 ml of fresh 1% HSA solution was added to the foam. After 30 minutes
the liquid was removed and 10 ml fresh 5% HSA solution was added so that
the foam was resuspended in solution. The liquid was quickly collected
from the bottom. The particles (microbubbles) had a diameter range of 1-12
microns with a wall thickness of 1-2 microns.
B. Gas Filled Microparticles
Encapsulated gas micropheres may be prepared according to WO-A-95/01187 by
mixing an aqueous solution of human serum albumin with a water insoluble
gas such as a perfluoroalkane (e.g. dodecafluoropentane).
EXAMPLE 4
Polymer Particles
A polymer particle suspension may be prepared by dissolving the
biodegradable polymer polyhydroxybutyrate-co-valerate in a suitable
organic solvent such as acetone, methylene chloride and the like,
precipitation in water and removal of the organic solvent by vacuum
distillation or diafiltration. Particle size may be selected to be within
the range 0.05 .mu.m to 10 .mu.m by choice of surfactant stabilizers, rate
of solvent evaporation, agitations as is well known in the art.
EXAMPLE 5
Optionally Photolabelled Nanoparticulate Suspensions
A solution of WIN 70177 (an iodinated contrast agent prepared according to
Example 24 below) and, optionally fluoroscein in the molar ratio 100:1,
optimally 50:1, most optimally 25:1, in DMSO (or DMF) is precipitated in
water. The resulting precipitate is milled as described in U.S. Pat. No.
5,145,684 together with a surfactant stabilizer (eg. Pluronic F108 or
Tetronic T-908 or 1508) to a particle size of 0.2 .mu.m and dispersed in
an aqueous medium to a contrast agent concentration of 0.5 to 25% by
weight and a surfactant content of 0.1 to 30% by weight. A cloud point
modifier such as polyethylene glycol 400 (PEG 400) or propylene glycol as
disclosed in U.S. Pat. No. 5,352,459 may also be included to ensure
stability on autoclave stabilization.
EXAMPLE 6
Photolabelled Nanoparticulate Suspensions
Phytochrome is added to an aqueous solution of sodium dodecyl sulphate
(pH>10). The resulting solution is added to a stirred solution of acetic
acid containing a surfactant (selected from PVP, pluronics and tetronics)
and the mixture is diafiltered to remove soluble salts, excess acid etc.
from the suspension yielding a dispersion of 10-100 nm particles.
EXAMPLE 7
Photolabelled Micelles
Indocyanine green (ICG) (0.1 to 10%) is mixed with 3% Pluronic F108 in
aqueous solution to form a micellar composition which is sterile filtered.
The ICG content used may be high (>0.5%) to produce mixed micelles or low
(<0.5%) to produce micellar solutions of ICG. ICG-concentrations of 0.2 to
0.5% are preferred.
EXAMPLE 8
Photo-labelled Liposomes
A liposome suspension is prepared using a 0.01 M solution of indocyanine
green and 5 to 10% of a phospholipid (10:1 ratio of lecithin to
dipalmitoylphosphatidyl serine). Preparation is effected by conventional
techniques (eg. ultrasound) followed by extrusion through controlled pore
size filters and diafiltration or microfluidisation. The resulting
liposomes are steam sterilizable and are sterile filterable and have
demonstrated physical stability under nitrogen for over six months.
EXAMPLE 9
Photo-labelled Emulsions
An oil in water emulsion is prepared from 10 g safflower oil, 10 g sesame
oil, 1.2 g egg phosphatides, 2.5 g glycerin, 0.5 to 10 g photo-label (eg.
fluorescein or indocyanine green) and water to 100 g total. Emulsification
is effected by conventional means and the resultant emulsion is sterile
filtered through 0.2 .mu.m sterile filters or steam sterilized using
conventional means.
EXAMPLE 10
Particulate Iodinated Compounds
WIN 70146 (an iodinated X-ray contrast agent prepared according to Example
23 below) was added to each of 3.times.1.5 oz brown glass bottles
containing approximately 12 ml of zirconium silicate, 1.1 mm diameter
beads in an amount sufficient to be 15% (wt/vol %) of the final
suspension. Bottle A was also made 3% (wt/vol %) Pluronic F-68 while
bottle B was made 3% (wt/vol %)) Pluronic F-108 and bottle C was made 3%
(wt/vol %) Tetronic T-908. The resulting suspensions were milled at approx
150 rpm for a total of 9 days with estimates of particle size determined
at various intervals as detailed below.
______________________________________
Average Particle Size
(nm)
Days of milling
F-68 F-108 T-908
______________________________________
2 1939* 158 162
3 223 161 162
7 157 158 156
9 158 159 159
After 1 week at room temperature
166 166 161
After autoclaving at 121 degrees C. for 20 min..sup.+
181 190 183
______________________________________
*Dioctylsulfosuccinate sodium (DOSS) was added at this point to aid in
milling in an amount equal to 0.2% (wt/vol %).
.sup.+ DOSS was added to the F108 and T908 samples for autoclaving as a
cloud point modifier (at 0.2%, wt/vol %).
These data demonstrate the unexpected ease of small particle preparation
with this agent (ie. WIN 70146) in both F108 as well as excellent
stability to heat (autoclaving) and time on the shelf.
EXAMPLE 11
Preparation and Acute Safety Testing of Nanoparticle Suspensions of WIN
70146 in Pluronic F108
WIN 70146 was prepared as in Example 10 and injected into the tail vein of
mice at doses of 3 ml/kg, 15 ml/kg, and 30 ml/kg (ie. 0.45 gm/kg, 2.25
gm/kg and 4.5 gm/kg). No untoward effects were noted in any of the mice at
any dose for a period of 7 days after which time the animals were
sacrificed. Gross observation of these animals did not reveal any obvious
lesions or disfigurations.
Further in depth safety studies in rats have not revealed significant
safety issues due to a single dose of WIN 70146/F108 at levels up to and
including 30 ml/kg (4.5 gm/kg). These studies included in-depth
histopathology, clinical chemistry, and in life observations.
EXAMPLE 12
Preparation of WIN 70146 in Pluronic F108 (I-404)
WIN 70146 was milled with 1.1 mm diameter zirconium silicate beads for 3
days under aseptic conditions. The concentration of this agent was 15% WIN
70146 in the presence of 4% Pluronic F-108. No additional salts or
surfactants were added. The average particle size of the resulting
nanoparticle suspension was 162 nm as determined by light scattering.
EXAMPLE 13
Preparation of an Autoclavable Formulation of WIN 70146 Using Pluronic
F-108 and PEG 400
WIN 70146 was milled with 1.1 mm diameter zirconium silicate beads in the
presence of Pluronic F-108 for 3 days. The final particle size was
determined to be 235 nm. At this point, sterile PEG 400 was added to the
suspension such that at completion, the formulation contained 15% (wt/vol
%) WIN 70146, 3% (wt/vol %) Pluronic F-108 and 10% PEG 400. This
formulation was then autoclaved under standard conditions (ie. 121 degrees
C. for 20 min.) resulting in a final particle size of 248 nm.
EXAMPLE 14
Demonstration of Light Scattering Above Incident Wavelengths of 600 nm by
Nanoparticle Suspensions of WIN 70146
A nanoparticle suspension of WIN 70146 was prepared as in Example 10 using
4.25% F108/10% PEG 400 which after autoclaving resulted in particles with
an average diameter of 228 nm. This suspension was then diluted in water
to various levels listed below. The per cent of incident light transmitted
was then determined for each suspension at several wavelengths (see
below). The suspensions were then dissolved by addition of methanol and
examined for per cent transmitted light against an equivalent solvent
blank. The results are given below.
______________________________________
Percent Transmission at 632 nm, 700 nm and 820 nm of
Both NanoParticulate WIN 70146 and Dissolved WIN 70146
Sample % T suspension % T solution
Conc 632 nm 700 nm 820 nm
632 nm
700 nm 820 nm
______________________________________
0.015% 54.7 64.5 77.0 100.4 100.3 100.5
0.0375%
25.4 36.6 53.8 99.9 99.9 99.9
0.075% 7.7 15.4 31.8 99.9 99.8 99.9
0.150% 0.5 1.9 8.6 41.4* 51.9* 66.2*
0.300% 0.0 0.1 0.8 1.2* 4.0* 13.5*
______________________________________
*These samples were not fully dissolved and showed visible turbidity
These results demonstrate that the suspensions are efficient light
scattering agents which do not absorb significant amounts of incident
light in these wavelength regions (ie., dissolved WIN 70146 does not
absorb light above 600 nm). Additional examination of the absorbance vs
wavelength for the dissolved agent does not show any evidence of light
absorbance from 600 to 800 nm while the nanoparticle agent shows a classic
absorbance decay due to scattering of the incident light.
EXAMPLE 15
Preparation of Nanoparticle Suspension of WIN 70177
A formulation of WIN 70177 (an iodinated X-ray contrast agent prepared
according to Example 24) was prepared as 15 gm of WIN 70177/100 ml of
suspension and 4.25 gm of Pluronic F108/100 ml of suspension and 10 gm of
PEG 400/100 ml of suspension. The suspension was milled for 5 days after
which the average particle size was determined by light scattering to be
about 235 nm. Stability testing in fresh rat plasma and simulated gastric
fluid did not show any aggregation.
EXAMPLE 16
Demonstration of Light Scattering above Incident Wavelengths of 600 nm by
Nanoparticulate WIN 70177
A nanoparticle suspension of WIN 70177 was prepared as in Example 15 using
4.25% F108/10% PEG 400 which after autoclaving resulted in particles with
an average diameter of 236 nm. This suspension was then diluted in water
to various levels listed below. The per cent of incident light transmitted
was then determined for each suspension at several wavelengths (see
below). The suspensions were then dissolved by addition of methanol and
examined for per cent transmitted light against an equivalent solvent
blank. The results are given below.
______________________________________
Percent Transmission at 632 nm and 700 nm of
Both Nanoparticulate WIN 70177 and Dissolved WIN 70177
Sample % T suspension % T solution
Conc 632 nm 700 nm 800 nm
632 nm
700 nm 800 nm
______________________________________
0.015% 53.3 62.8 73.1 102.2 101.9 101.8
0.0375%
34.6 45.7 59.1 102.3 101.9 101.8
0.075% 25.8 36.8 51.1 100.9 100.8 101.0
0.150% 6.7 13.6 26.3 59.5* 67.8* 77.0*
0.300% 0.1 0.6 3.2 7.4* 14.4* 26.8*
______________________________________
*Did not fully dissolve; particles still present.
These data demonstrate the scattering abilities of the particulate form of
WIN 70177 while the dissolved material does not absorb any energy over the
wavelength of light examined. Further, an examination of the absorbance
due to the particulate WIN 70177 and that due to the dissolved WIN 70177
shows that the particulate material provides an exponential drop in
absorbance with wavelength as would be expected for scattering due to
suspended particles while the soluble material has virtually no absorbance
at all even at 5 times the concentration.
EXAMPLE 17
Preparation of a Nanoparticle Suspension of WIN 67722
A formulation of WIN 67722 (an iodinated X-ray contrast agent as described
in U.S. Pat. No. 5,322,679) was prepared as in Example 1 using 3% Pluronic
F108 and 15% PEG 1450. The suspension was milled for 3 days and achieved a
particle size of 213 nm (small fraction at 537 nm) as determined by light
scattering with a Coulter N4MD particle sizer.
EXAMPLE 18
Demonstration of Light Scattering above Incident Wavelengths of 600 nm by
Nanoparticulate WIN 67722
A nanoparticle suspension of WIN 67722 was prepared as in Example 17 using
3% Pluronic F108 and 15% PEG 1450 which after autoclaving gave particles
with an average diameter of 214 nm. This suspension was then diluted in
water to various levels listed below. The per cent of incident light
transmitted was then determined for each suspension at several wavelengths
(see below). The suspensions were then dissolved by addition of methanol
and examined for per cent transmitted light against an equivalent solvent
blank. The results are given below.
______________________________________
Percent Transmission at 632 nm and 700 nm of
Both NanoParticulate WIN 67722 and Dissolved WIN 67722
Sample % T suspension % T solution
Conc 632 nm 700 nm 820 nm
632 nm
700 nm 820 nm
______________________________________
0.015% 47.9 57.1 69.2 99.9 99.9 100.6
0.0375%
20.5 29.9 45.6 100.2 100.2 100.4
0.075% 4.8 9.9 22.1 100.1 100.2 100.4
0.150% 0.2 1.0 4.9 48.2* 55.3* 65.5*
0.300% 0.0 0.0 0.2 1.3* 35* 10.7*
______________________________________
*Did not fully dissolve; particles still present
These data demonstrate the scattering abilities of the particulate form of
WIN 67722 while the dissolved material does not absorb any energy over the
wavelength of light examined. Further, an examination of the absorbance
due to the particulate WIN 67722 and that due to the dissolved WIN 67722
shows that the particulate material provides an exponential drop in
absorbance with wavelength as would be expected for scattering due to
suspended particles while the soluble material has virtually no absorbance
at all even at 5 times the concentration.
EXAMPLE 19
Preparation of Nanovarticle Suspension of WIN 72115
Nanoparticle WIN 72115 (a fluorescent iodinated contrast agent as described
in Example 21 below) was prepared by combining WIN 72115 and Pluronic F108
(BASF, Parsippany, N.J.) in a glass jar at concentrations of 15 gm/100 ml
suspension and 3 gm/100 ml suspension. The jar was then half filled with
1.0 mm diameter zirconium silicate beads and sufficient water added to
complete the required concentrations of agent/surfactant as noted above.
Alternatively, the surfactant can be dissolved in the water before
addition to the jar (with or without sterile filtration through 0.2 micron
filters).
The jar is then rolled on its side for not less than 24 hours or more than
14 days at a rate of rotation sufficient to cause the beads within the jar
to "cascade" down the walls of the jar as it turns (see U.S. Pat. No.
5,145,684). At the end of the milling cycle, the material is harvested
from the jar and separated from the milling beads.
Nanoparticles of WIN 72115 prepared in this manner have an average particle
size of 225 nm by light scattering.
WIN 72115 was designed to be excited with incident radiation from an Argon
Ion laser (in the green, near 514 nm) and emit light at wavelengths above
that value. Thus, after injection, illumination of the patient with green
light would stimulate emission of light of a slightly different wavelength
that could be used for diagnostic purposes. The key features of this agent
are that it can be prepared as nanoparticles, remain within the
vasculature for greater than 15 minutes, provide both scattering and
fluorescence contrast for light imaging.
In place of WIN 72115, the photolabelled agent of Example 22 below may be
used.
EXAMPLE 20
Light Scattering from Polymeric Particles--Dependence Upon Particle Size
and Concentration
Three samples of polystyrene latex particles were diluted to various
extents and examined for their effects upon transmitted light at several
different wavelengths. The results confirm that larger particles and
higher concentrations result in better scattering of the incident light.
______________________________________
concentration
Per cent Transmission
Sample (Wt/vol %) 600 nm 700 nm
820 nm
______________________________________
170 nm .0025 97.9 98.3 98.7
.025 94.8 96.3 97.4
.075 89.3 92.8 95.2
300 nm .0025 99.3 99.5 99.6
.025 92.4 94.5 95.8
.075 83.1 88.3 91.8
500 nm .0025 98.8 99.1 99.4
.025 88.1 91.4 93.9
.075 68.3 76.5 83.0
______________________________________
EXAMPLE 21
3-(N-Acetyl-N-ethylamino)-5-[(5-dimethylamino-1-naphthylsulfonyl)aminol-2,4
,6-triiodobenzoic Acid Ethyl Ester (WIN 72115)
To a stirred solution of ethyl
3-(N-acetyl-N-ethylamino)-5-amino]-2,4,6-triiodobenzoate (11.6 g, 18.5
mmol) in pyridine (75 ml) cooled in ice bath is added 60% NaH/oil
dispersion (1.8 g, 46.3 mmol). After the reaction of NaH with the amino
group is over, dansyl chloride (5 g, 18.8 mmol) is added. The resulting
reaction mixture is stirred in ice bath for 4 hours and at room
temperature for 20 hours. After quenching with acetic acid (10 ml), the
brown solution is concentrated on a rotary evaporator. The brown residue
is first washed with hexanes and then slurried in water (200 ml). The
resulting dirty yellow gummy solid is collected, washed with water, dried,
and recrystallized from ethanol to provide 5.3 g (33%) of bright yellow
crystals: mp 238-240.degree. C., ms (FAB) 862 (90%, MH). Anal. Calcd. for
C.sub.25 H.sub.26 I.sub.3 N.sub.3 O.sub.5 S: C, 34.86; H, 3.05; N, 4.88;
I, 44.20. Found: C, 34.91; H, 3.02; N, 4.74; I, 44.53. .sup.1 H-NMR and
.sup.13 C-NMR spectra are consistent with the structure:
##STR1##
EXAMPLE 22
2-(3,5-Bisacetylamino-2,4,6-triiodobenzoyloxy)ethyl
N-Fluoreceinylthiocarbamate
A mixture of 2-hydroxyethyl 3,5-(bisacetylamino)-2,4,6-triiodobenzoate
(0.658 g, 1 mmol), fluorecein isothiocynate (0.389 g, 1 mmol), 60% NaH/oil
dispersion (0.24 g, 6 mmol) and DMF (25 ml) is stirred at ambient
temperature for 26 hours and then quenched with 6N HCl (2.5 ml). The
resulting mixture is concentrated on a rotary evaporator under reduced
pressure. The yellow solid residue is washed with water and recrystallized
from DMF to yield yellow crystals of the product in 65% yield. Elemental
analysis and spectral data are consistent with the structure:
##STR2##
EXAMPLE 23
Benzoic acid, 3.5-bis(acetylamino)-2.4.6-triiodo-1-(ethoxycarbonyl)pentyl
ester (WIN 70146)
To a stirred solution of sodium diatrizoate (150 g, 235.2 mmole) in dry DMF
(1200 ml) at room temperature, was added ethyl 2-bromohexanoate (63.8 g,
285.8 mmole, 1.09 eq.). The solution was heated overnight at 90.degree.
C., then cooled to 60.degree. C. The reaction mixture was then poured into
201 of water with stirring. The resulting white precipitate was collected
by filtration and dried at 90.degree. C. under high vacuum. The crude
material was recrystallized from DMF/water to give, after drying,
analytically pure product; mp 263-265.degree. C. The MS and .sup.1 H-NMR
(300 MHz) spectral data were consistent with the desired structure.
Calculated for C.sub.19 H.sub.23 I.sub.3 N.sub.2 O.sub.6 : C, 30.15; H,
3.04; N, 3.70; I, 50.35. Found: C, 30.22; H, 3.00; N, 3.66; I, 50.19.
EXAMPLE 24
Propanedioic acid,
[[3,5-bis(acetylamino)-2,4,6-triiodobenzoyl]oxy]methyl-bis(1-methylethyl)e
ster (WIN 70177)
To a stirred mixture of sodium diatrizoate (393 g, 616 mmole) in 500 ml of
DMSO at room temperature, was added 173 g (616 mmol) of diisopropyl
2-bromo-2-methylmalonate and the solution was heated at 90-100.degree. C.
under an atmosphere of argon for 56 hours. After cooling, the solution was
slowly added to 101 of water with mechanical overhead stirring. The
precipitated solid was allowed to settle for 6 hours and then collected by
filtration. The crude product was washed thoroughly with water (41) and
dried at room temperature overnight. The solid was digested with a
solution of potassium bicarbonate (3 g in 700 ml of water containing 15 ml
of isopropanol), water and then air dried for 12 hours. Recrystalization
from DMF followed by washing with water and drying under high vacuum gave
255 g (51%) of analytically pure product; mp 258-260.degree. C. The MS and
.sup.1 H-NMR (300 MHz) spectral data were consistent with the desired
structure.
Calculated for C.sub.21 H.sub.25 I.sub.3 N.sub.2 O.sub.8 : C, 30.98; H,
3.10; N, 3.44; I, 46.76. Found: C, 30.96; H, 3.00; N, 3.44; I, 46.77.
EXAMPLE 25
In vivo Light Imaging Studies
A. Particulate Scattering Agents
A suspension of multilamellar liposomes formed in a solution of 40% (wt/vol
%) iodixanol were injected into white rats which had been implanted with a
hepatoma 9L tumor on their rear flank. The injection was imaged using a
time gated diode laser incident at 780 nm with detection of the scattering
component at 180 degrees to the incident light using fiber optic cables
and a phase sensitive detection device in the laboratory of Dr. Britton
Chance at the University of Pennsylvania. The liposome particles enhanced
scattering in the tumor over the background signal by more than 4.times.
at the dose administered (i.e. 3 ml/kg). While not optimized, these data
indicate the feasibility of contrast by scattering agents for light
imaging.
B. Fluorescent particles for light imaging contrast
A suspension of liposomes were prepared in the presence of 0.7
micrograms/ml of indocyanine green (ICG) and sterilized using steam and
pressure. The resulting particles had an average diameter of approximately
120 nm as determined by light scattering using a Horiba 910 particle
sizing instrument. Upon injection into the rat flank tumor model, these
liposomes afforded significantly longer residence in the tumor of the
fluorescent agent (i.e. the ICG) than observed with a homogeneous solution
of ICG alone. This is useful for imaging in that signal averaging
techniques can be applied to enhance the image as well as to mark sites of
leaky vasculature. These studies were also carried out at the University
of Pennsylvania in the laboratory of Dr. Britton Chance.
EXAMPLE 26
Use of Contrast Media for Enhancement of Laser Doppler Measurement of Blood
Flow in the Skin
Approximately 0.5 to 1 hour before the measurements are to be made, a
sterile aqueous suspension containing 5-20 mg of suspended particles of a
dye (e.g. 3,3'-diethylthiatricarbocyanine iodide) with an absorbing
maximum between 600 and 1300 nm is administrated by intravenous injection.
The mean particle size is preferably about 800 nm and as suspension medium
is preferably used physiological saline.
The measurement of blood flow is made after the concentration of contrast
agent particles in the blood has stabilized. Measurement may be made with
a standard laser Doppler instrument, for example that from Lisca
Development AB, Kinkoping, Sweden, that optionally may be modified to
incorporate a laser source operating at 830 or 780 nm (see Abbot et al.,
J. Invest. Dermatol., 107: 882-886 (1996)).
EXAMPLE 27
Use of Contrast Media for Enhancement of Measurement of Blood Flow through
the Skin with Confocal Microscopy
Approximately 0.5 to 1 hour before the measurements are to be made, a
sterile aqueous suspension containing 5-20 mg of dye (e.g.
3,3'-diethylthiatricarbocyanine iodide) with an absorbing maximum between
600 and 1300 nm is administrated by intravenous injection. The mean
particle size is preferably about 800 nm and as suspension medium is
preferably used physiological saline.
The measurement of blood flow is made by following the movement of the
particles through the capillaries with the confocal microscope.
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